Project 2. The Arbovirus Dilemma: How do arthropod-borne viruses counteract the immune defenses of both host and vectors?
Arthropod-borne viruses (arboviruses) face a two-fold challenge in countering the antiviral defenses of both vertebrate hosts and arthropod vectors. In vertebrates, the front-line defense against viruses is the interferon (IFN) response, whereas in many arthropods, it is RNA interference (RNAi), the targeted destruction of messenger-sense RNA with homology to a double-stranded RNA trigger. While the strategies used by arboviruses to elude the IFN response have been studied extensively, almost nothing is known about the mechanisms employed to counteract RNAi. Infection by mosquito-borne dengue virus triggers RNAi in Aedes aegypti and Drosophila melanogaster, and this response significantly reduces virus replication.
Given that dengue establishes a persistent infection in its mosquito vector, these findings suggest that it must elude RNAi control via production of a viral supressor of RNAi, or, alternatively, via replication in protected sites within the cell. We are currently testing the hypothesis that dengue virus produces a single protein that serves to suppress both IFN and RNAi.
Funding for this study is provided by NIH 1R21AI082399-01
Due to their high rates of mutation, large population sizes, and rapid rates of replication, RNA virus populations comprise a "quasispecies", or swarm of mutant genomes. The diversity of a quasispecies influences viral adaptation, disease severity, and response to antiviral therapies. Identifying the mechanisms that shape quasispecies diversity is necessary to better prevent virus emergence and control virus disease, particularly for arthropod-borne viruses (arboviruses), all of which are RNA viruses and many of which are significant emerging threats to global public health. Previous studies have revealed that arbovirus quasispecies diversity may differ between vertebrate hosts and arthropod vectors.
It has been shown that quasispecies diversity of West Nile virus is significantly greater in mosquitoes than in birds, suggesting that the antiviral defenses of mosquitoes select for virus diversification. the recent discovery of RNA interference (RNAi), the targeted destruction of RNA with high homology to a double-stranded RNA trigger, has revealed a general pathway by which arthropods regulate viral replication. In collaboration with Greg Ebel and Doug Brackney at UNM, this project tests the hypothesis that the RNAi response of vectors imposes selection for mutations that enable virus populations to escape RNAi and RNAi thereby drives diversification of viral quasispecies. To do so we are monitoring the evolutionary responses of two emerging arboviruses, dengue virus and West Nile virus to downregulation of RNAi.
Project 4: Competition among dengue serotypes, ecotypes and strains
Competition between two strains of a single pathogen can prevent disease emergence if an established human strain excludes invasion of a novel strain from a zoonotic reservoir. Alternatively competition can facilitate resurgence if a highly virulent strain displaces one that is weakly virulent. The four serotypes of mosquito-borne dengue virus emerged independently from a sylvatic, zoonotic cycle into an endemic cycle between humans and peridomestic Aedes. Subsequently the endemic ecotypes have diversified into separate subtypes. We have investigated competition (i) between sylvatic and endemic ecotypes of DENV and (ii) between highly and lowly virulent subtypes of endemic DENV serotype 3 (DENV-3) in mosquito cells in culture and in vivo. We continue to pursue these studies and to investigate the mechanisms for competition, particularly the role of host defenses in mediating competition.
Funding for this study was provided by NIH 2P20RR016480-06 NIH NM Ideas Network for Biomedical Research
Hanley, K.A., J.T. Nelson, E. E. Schirtzinger, S.S. Whitehead, C.T. Hanson. 2008. Superior infectivity for mosquito vectors contributes to competitive displacement among strains of dengue virus. Biomed Central Ecology 8:1.
Pepin, K.M., K. Lambeth, K.A. Hanley. 2008. Asymmetric competitive suppression between strains of dengue virus. Biomed Central Microbiology 8:28.
Migratory birds are the major reservior of influenza A virus (AIV) and a source of pandemic strains in humans. Surveillance of AIV in wild birds is a critical foundation for efforts to predict and control AIV emergence. The Bosque del Apache (BdelA) National Wildlife Refuge, which is surrounded by desert, represents a geographic bottleneck where southbound migratory waterfowl converge. In conjunction with collaborators Jeanne Fair and MArk Jankowski at the Los Alamos National Labs, Tim Wright at NMSU and investigators at BdelA, we are surveying target waterfowl species for AIV using RT-PCR on oropharyngeal and cloacal swabs. These efforts are linked to an ongoing effort, also involving Laura Boucheron at the NMSU Elctrical and Computer Engineering Department to develop and deploy GPS-based tracking devices on waterfowl to better understand the potential movement of AIV into and out of New Mexico.
Project 6: Novel synergies for antiviral drug design
The genus Flavivirus, comprising 80 species of single-stranded, positive-sense RNA viruses, includes a large number of globally significant emerging pathogens. In the last 50 years many flaviviruses, such as dengue, West Nile, and tick-borne encephalitis viruses, have exhibited dramatic increases in incidence, disease severity and/or geographic range. For example the annual number of cases of dengue hemorrhagic fever cases worldwide has risen from nearly 0 in the 1950’s to 500,000 today. These trends are exacerbated by failure of vector control to limit virus spread as well as the absence of vaccines for viruses such as dengue and West Nile. Thus effective antiviral therapies are urgently needed to ameliorate the disease burden imposed by flaviviruses. At present, however, no licensed therapies are available for any flavivirus, largely because existing broad-spectrum drugs have failed to show efficacy against flaviviruses or have generated unacceptably high levels of toxicity.
To overcome these limitations, it will be necessary to develop not only new antiviral drugs but also innovative strategies to lower their effective dose and thereby mitigate toxicity. The recent discovery that certain FDA-approved fluoroquinolone antibiotics enhance the activity of the RNA interference pathway suggests one promising new strategy. RNA interference is a ubiquitious antiviral defense of eukaryotes that acts by targeting small interfering RNA’s (siRNA’s) to complementary regions in the viral genome; genomes bound to siRNA’s are marked for cleavage and degradation. Because binding of siRNA’s to a target sequence requires nearly perfect complementarity, RNAi imposes selection pressure for viral mutation that disrupts such complementarity. The proposed research will test the hypothesis that enhancing RNAi will amplify the effect of mutagenic nucleoside analogs on dengue virus mutation rate and replication, and that the synergistic effect of RNAi enhancement will accelerate lethal mutagenesis driven by nucleoside analogs. If this hypothesis is correct, the finding of this study will represent a significant advance in the development of new therapies for flaviviral disease.